Abstract

Genetic studies of organisms based on coalescent modeling and paleoenvironmental data,
including a new study in BMC Biology of Mexican jays in the sky islands of Arizona and northern Mexico, show that populations
differentiated in multiple refugia during and after glacial cycles.

Minireview

The general cooling of the world's climate that began in the Tertiary and culminated
in the Pleistocene glacial cycles from about 2.4 million years ago attracted the attention
of evolutionary biologists because of its possible effect in changing species distributions,
and thus on the speciation of organisms. The role of these climatic fluctuations on
speciation has been much debated. At one end of the debate, some researchers argued
that the cooling suppressed or slowed speciation, as leading-edge waves of species
populations repeatedly colonized deglaciated regions in the interglacial periods [1,2]. This form of repeated colonization of genetically similar individuals from the same
source populations can prevent genetic differentiation required for speciation. Others
thought that the cooling, and the barriers of ice that divided up populations, increased
the rate of speciation; in an extreme example of this view, Ernst Mayr wrote in his
classic 1970 book [3] that "Evolutionists agree on the overwhelming importance of Pleistocene barriers
in the speciation of temperate zone animals".

Data from studies of North American songbirds have been useful in showing which of
these two views is correct. As late as 1999, it was thought that species and species
complexes of North American songbirds diverged in the late Pleistocene, which would
support the view that climate cooling increased the rate of speciation [4]. This was, however, refuted convincingly by mitochondrial DNA data that suggested
that the emergence of new songbird species appeared repeatedly over the past 5 million
years, which would mean a much smaller role for climate cooling in speciation [5].

The current consensus is that some species of songbirds originated earlier in the
Pleistocene, before the glaciations started [5-7]. It is also generally agreed that strong population structure has evolved in songbirds
and in many other organisms [5-8]. When many genetic differences accumulate in different populations, this structures
species into isolates that can be a precursor to speciation. However, there is some
evidence that songbird speciation might have been completed during late glacial advances
by repeated bouts of geographical isolation, as shown by the fact that divergence
times estimated with a molecular clock in superspecies complexes of boreal (boreal
forest) superspecies of North American birds date to the Pleistocene [9]. These complexes are groups of very similar emergent species with adjacent distributions
that are restricted to boreal forests that were glaciated in the Pleistocene.

Uncertainty in inferences of glacial refugia

Although there is compelling evidence that ancestral source populations can differ
genetically, there is uncertainty about whether isolation of populations that survived
and differentiated in glaciated areas called glacial refugia is required to explain
genetic differentiation in extant populations [7,10]. Furthermore, inference of the number of these refugia and the timing of isolation
of populations has, until recently, depended on the construction of gene trees, assumptions
about whether these trees reflect population trees, calibrations of molecular clocks
and mutation rates of the genes being studied. All these components have uncertainties
inherent in their estimates. Innovative new studies have, however, begun to address
these uncertainties with exciting insights into the impact of Pleistocene climatic
cycles on population differentiation and, potentially, on speciation [10-14].

Evidence for divergence within species complexes of songbirds in both the Pleistocene
period and postglacially has been presented in recent studies [13,14]. The yellow-rumped warbler complex comprises two North American migratory subspecies,
the myrtle warbler (Dendroica coronata coronata and Audubon's warbler (D. c. auduboni), previously thought to be separate species, and two largely sedentary (non-migrating)
forms from Mexico (D. c. nigrifrons) and Guatemala (D. c. goldmani). The North American forms breed in higher-latitude locations than the Mesoamerican
forms, locations that were glaciated in the past. The North American forms hybridize
with the Mesoamerican forms only in a narrow hybrid zone in British Columbia and Alberta,
but they migrate and overlap with the Mesoamerican forms in winter.

Phylogenetic analyses of three mitochondrial DNA genes using Bayesian methods that
account for phylogenetic uncertainty have shown, surprisingly, that the two Mesoamerican
forms are reciprocally monophyletic, that is, that they each form a monophyletic group
that is phylogenetically separated from the other, whereas the North American forms
have high levels of shared ancestral polymorphisms [13]. Assuming a mutation rate of 2% per million years, a coalescent approach yielded
population divergence times of about 400,000 years ago between Mesoamerican and North
American forms and 16,000 years ago between the two North American forms. Coalescent
theory is a population genetics model that traces all the alleles of a gene in a population
sample to one ancestral copy shared by all members of the population, which is called
the most recent common ancestor (MRCA). By applying a mutation rate for the gene it
is possible to obtain the time in years when the MRCA existed, which approximates
when the forms diverged unless they continued to exchange alleles for some time after
they separated. However, when dated with a wide range of gene-specific mutation rates,
the uncertainty in dates was revealed, ranging up to 1.9 million years ago between
migratory and sedentary forms and up to 41,000 years ago between migratory forms.

Coupling paleoenvironmental and genetic modeling

With such imprecision in estimating divergence times, it is difficult to test hypotheses
of postglacial population differentiation or rapid speciation using genetic data alone.
Now, however, fossil paleoecological data have emerged that can provide an independent
timeframe for recent postglacial genetic divergence. McCormack et al. in a recent study in BMC Biology [14] capitalized on populations of Mexican jays (Aphelocoma ultramarina) in the 'sky islands' – isolated mountain niches – of southwestern USA and northern
Mexico; these birds are ecologically tied to pine-oak woodlands (Figure 1). Fossilized plant material in the garbage collected in the middens of packrats (Neotoma spp.) showed that the sky islands were connected by continuous woodlands 18,000 years
ago, at the last glacial maximum, but as climate warmed in the past 9,000 years the
woodlands have been driven to higher elevations and have been displaced by grassland
and desert at lower elevations. The authors [14] therefore predicted that populations of jays should share common alleles from the
ancestral population, but that each population should have a suite of 'private' alleles
that has accumulated by mutation in the postglacial period. That is exactly what they
found in judiciously chosen mitochondrial and nuclear loci with high mutation rates.

Figure 1. The Mexican jay is a sedentary species found in pine-oak woodlands in the sky islands
in the southwestern USA and northern Mexico. Different populations have differentiated
genetically within the last 10,000 years. Photo by TJ Ulrich with permission from
Visual Resources for Ornithology, the Academy of Natural Sciences, Philadelphia, PA.

McCormack et al. then subjected the genetic data for selected population pairs to a multilocus coalescent
analysis to estimate the time of population divergence and obtained confirmation of
postglacial differentiation in the past 10,000 years or less, on the basis of the
90% highest posterior density distributions. By fitting a model of population splitting
to explain the genetic data it is possible to generate a large number of possible
estimates of a parameter, which forms the posterior density distribution of parameters,
such as population divergence time. This method also takes into account the uncertainties
in the simulation process. Additional corroboration of the coalescent estimates was
obtained from genetic distances corrected for within-species polymorphism, with the
exception that divergence times in the western sky islands in the Arizona 'archipelago'
were found using this method to range up to 81,000 years ago. The general message
that emerges from this excellent study [14] is that detection of postglacial divergence requires large sample sizes to detect
private alleles arising from new mutations and to reduce stochasticity in the coalescent
process modeled with or without migration.

Ecological-niche modeling and statistical testing of hypotheses

Other exciting developments that are helping us to understand the impact of climate-induced
shifts in the Pleistocene on distribution of populations, and thus on speciation,
include the use of ecological-niche modeling to predict past geographic distributions
of ancestral source populations. This innovative approach provides the tools for statistical
testing of hypotheses about multiple refugia by integrating inferred past distributions
with coalescent-based genetic models [10-12]. Again, these studies are using the multiple replicates provided by different sky-island
populations in North America and include a plant-insect herbivore association [12] and montane grasshoppers [10,11].

Cutting-edge research from the Knowles laboratory at the University of Michigan [10,11] using ecological-niche modeling has provided a reconstructed historical distribution
of the flightless montane grasshopper (Melanoplus marshalli), revealing that, during glacial maxima, sky-island grasshopper populations in Colorado
and Utah must have been displaced to lower refugial areas nearby. By coupling this
approach with genetic modeling, the authors were able to test statistically whether
the grasshoppers survived in a single ancestral refugial population or multiple refugial
populations. Genetic modeling in a coalescent framework not only accounts for the
stochastic effects of genetic drift in patterns of population divergence, but by simulating
DNA sequences it also incorporates the effect of mutational variance. This makes it
possible to use the amount of lineage sorting in extant populations, as measured by
the number of deep coalescents in gene trees, to test whether the amount of discord
between the sequence data and a two-refugia model is significantly lower than expected
under a single refugium model. Recolonization from multiple or single refugia in interglacials
could therefore possibly explain why populations of grasshoppers have either evolved
strong geographic structure or have speciated, whereas others have differentiated
relatively little.

By bringing more biological realism from the natural history of organisms into ecological
and genetic modeling of population divergence, the impact of glacial cycles on current
biodiversity is being revealed in increasing detail. An interesting aspect of several
of these studies is that they often choose to sequence the mitochondrial cytochrome
oxidase gene (COI), sometimes in tandem with multiple nuclear genes. COI is used because it has sufficient variable sites in the part of the gene used in DNA
barcoding studies to provide sufficient resolution for coalescent analysis. This point
is made clearly in the Mexican jay study [14] and is a straightforward prediction of the faster coalescent times and resolving
power of mitochondrial genes [15]. Although the current emphasis in detecting very recent (postglacial) population
divergence is on analysis of increasing numbers of nuclear sequences to reduce variance
across loci, it seems unwise not to combine these with one or more faster evolving
mitochondrial genes, as was done so effectively with the montane grasshoppers [10]. Ultimately, such a unified approach is likely to help delimit species genetically
and to connect the processes of population divergence and species recognition in a
more rigorous way.

Acknowledgements

I thank Visual Resources for Ornithology for permission to reproduce Figure 1.